Pain is a subjective experience that arises from neural responses to noxious stimuli in multiple brain regions and has both discriminative and emotional components. Pain plays a crucial, protective role in preserving bodily homeostasis by triggering a reflex, such as withdrawal, to reduce exposure to harmful stimuli. However, pain processing can become abnormal, resulting in chronic pathological conditions. Pathological chronic pain is not only a physical ordeal, but also an emotional drain, and is refractory to currently available treatments. Thus, understanding the neural pathways that process the emotional, affective, and autonomous responses to painful stimuli is essential in forming the new foundations for understanding and treating pathological chronic pains. It is known that the emotional and affective pain pathway involves the parabrachial nucleus (PB) located in the brainstem, which is subdivided into two major divisions: lateral PB (PB-l) and medial PB (PB- m). Noxious information is relayed through PB to the limbic system to generate affective and autonomous responses. However, due to the lack of molecular tools, it had not been possible to study the precise connectivity and functions of different PB neurons activated by selected painful stimuli. In this proposal, I am using a highly innovative novel technology recently developed in our lab to investigate specifically the PB neural circuits involved in processing noxious thermal (orofacial-) pain in mouse. The hypothesis that I am going to test is: thermal nociceptive PB-l and PB-m neuronal ensembles are embedded in different sub-circuits, have different properties and different functions in regulating affective thermal (orofacial) pain perception. Specifically, using the novel technique, I will express fluorescent proteins or optogenetic tools in PB neurons activated by capsaicin, a surrogate for noxious heat stimulus. I will identify the axonal projection targets and certain inputs of thermal nociceptive PB-l and PB-m neurons. I will examine the neurochemical and electrophysiological properties as well as synaptic connectivity of capsaicin- activated PB neurons with slice electrophysiology. Finally, I will use optogenetic activation and inhibition to determine the functions of capsaicin-activated PB neurons in noxious heat pain perception using an operant orofacial pain behavior. These studies are expected to fill the knowledge gap of the PB neural pathways, and provide new insights into the circuit mechanism that process the emotional, affective, and autonomous responses to painful stimuli.